[show abstract][hide abstract] ABSTRACT: Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.
Molecular and Cellular Biology 11/2000; 20(20):7463-79. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lysophosphatidic acid (LPA) in biological fluids binds to serum albumin and other proteins that enhance its effects on cellular functions. The actin-severing protein gelsolin binds LPA with an affinity (K(d) = 6 nm) similar to that of the G protein-coupled LPA receptors encoded by endothelial differentiation genes 2, 4, and 7 (Edg-2, -4, and -7 receptors) and greater than that of serum albumin (K(d) = 360 nm). At concentrations of 10% or less of that in plasma, which are observed in fluids of injured tissues, purified and recombinant gelsolin augment LPA stimulation of nuclear signals and protein synthesis in rat cardiac myocytes (RCMs) that express Edg-2 and -4 receptors. At concentrations of 20% or more of that in plasma, gelsolin suppresses LPA stimulation of RCMs. The lack of effect of gelsolin on RCM responses to monoclonal anti-Edg-4 receptor antibody plus a phorbol ester without LPA attests to its specificity for LPA delivery and the absence of post-receptor effects. Inhibition of gelsolin binding and cellular delivery of LPA by l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP2) and peptides constituting the two PIP2 binding domains of gelsolin suggests competition between LPA and PIP2 for the same sites. Thus, delivery of LPA to RCMs is affinity-coupled to Edg receptors by gelsolin in a PIP2-regulated process.
Journal of Biological Chemistry 06/2000; 275(19):14573-8. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glima 38 is an N-glycated neuroendocrine membrane protein of M(r) 38,000, which is recognised by autoantibodies in approximately 20% of patients with Type I (insulin-dependent) diabetes mellitus. The aim of this study was to characterise the carbohydrate moiety and generate peptide maps of glima 38.
Sera of high immunoreactivity to glima 38 were used to isolate 35-S methionine-labelled protein from betaTC-3 cells and a neuronal cell line GT1.7. Tunicamycin was used to inhibit N-glycation of glima 38 and define the core protein. The carbohydrate moiety was characterised for tunicamycin sensitivity, lectin binding and susceptibility to different endoglycosidases. The protein moiety was subjected to digestion by proteases to define peptide maps.
The autoreactive epitopes in glima 38 recognised by Type I diabetic sera are conformational and independent of the carbohydrate moiety. Inhibition of N-glycation of glima 38 in vivo, shows a protein core of M(r) 22,000 in both pancreatic beta-(betaTC3) and neuronal (GT1.7) cell lines. The carbohydrate moieties in the two cell types are distinct but contain a similar amount of terminal sialic acid residues and at least five oligosaccharide chains Glima 38 binds Triticum vulgare and Ricinus communis I lectins. Endoproteinase treatment of the M(r) 22,000 core protein results in peptides of M(r) 4500 and M(r) 20,000 with Lys-C, and peptides of M(r) 4000 and M(r) 11,000-12,000 with Glu-C/V8 and Asp-N proteases.
The biochemical properties of glima 38 define it as a new autoantigen in Type I diabetes and provide a basis for its purification.
[show abstract][hide abstract] ABSTRACT: Kidney cells, especially the epithelial cells lining the collecting tubules in the inner medulla, are constantly exposed to concentrated urine. They are protected from the osmotic effect of high levels of sodium ion and urea by accumulating compatible osmolytes such as sorbitol, betaine, and myo-inositol. These osmolytes are involved in maintaining cell volume and electrolyte contents because they do not perturb the protein structure and function over a wide range of concentrations. Sorbitol is produced via the reduction of glucose by aldose reductase (AR), while betaine and myo-inositol are transported into the cells through specific transporters. Under hyperosmotic stress, transcriptions of genes encoding these proteins are highly induced. The induction of transcription was found to be mediated through the osmotic response elements (OREs) located in the 5' flanking sequences of these genes. We had earlier identified the OREs in human AR gene. In this study we purified and identified the osmotic response element binding protein (OREBP). OREBP is a transcription factor of approximately 200 kDa in size, characterized by a Rel-like DNA binding domain and a glutamine-rich transactivation domain. Dominant negative OREBP significantly diminished hyperosmotic AR gene induction. Immunohistochemical analysis showed that this transcription factor is rapidly translocated into the nucleus upon hyperosmotic stress.
Biochemical and Biophysical Research Communications 05/2000; 270(1):52-61. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.
Molecular and Cellular Biology 05/2000; 20(7):2488-97. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.
The Journal of Cell Biology 03/2000; 148(3):427-40. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.
[show abstract][hide abstract] ABSTRACT: Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 and 170 kD) from Dictyostelium extracts that independently reconstitute plus-end-directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245-kD protein (DdUnc104) is a close relative of Caenorhabditis elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily, which are monomeric.
The Journal of Cell Biology 12/1999; 147(3):493-506. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bronchiolitis obliterans occurs in 30% to 80% of lung-transplant recipients and is a direct cause of death in more than 40% of patients with this complication. This study assessed the potential utility of measuring fibroblast-proliferative activity in bronchoalveolar lavage fluid from lung-transplant recipients to better understand the pathogenesis of this process.
The capacity of bronchoalveolar lavage fluid obtained from transplant recipients, during routine surveillance bronchoscopy, to stimulate the proliferation of human lung fibroblasts in vitro was assessed retrospectively and compared to that of control subjects. For each recipient, a correlation was made between the fibroblast-proliferative activity in serial lavage samples over time and the other modalities employed for detecting post-transplant complications including spirometry, transbronchial lung biopsy, and high-resolution computed tomography.
There was a significant difference in fibroblast-proliferative activity between volunteer and transplant recipient groups (p = 0.002). Further, for each transplant recipient, the decline in the forced expired flow rate between 25% and 75% of expired volume (FEF(25%-75%)) was correlated with the mean fibroblast-proliferative activity during the period of this study (r = 0.83; p = 0.04).
A sustained increase in fibroblast-proliferative activity in lavage supernatant precedes both histologic and physiologic evidence of bronchiolitis obliterans. Relative to an increase in fibroblast-proliferative activity or abnormalities in FEF25%-75%, a decrease in forced expiratory volume in 1 second is a late finding.
The Journal of Heart and Lung Transplantation 11/1999; 18(10):972-85. · 5.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.
Protein Science 11/1999; 8(10):2177-85. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prostaglandin E2 (PGE2) is a potent mediator generated in immune tissues by cyclooxygenation of arachidonic acid. PGE2 affects T cell functions through four homologous G protein-coupled receptors termed EP1R, EP2R, EP3R, and EP4R that differ in tissue distribution and signaling. Antigen-evoked secretion of interferon-gamma (IFN-gamma) by sperm whale myoglobin-specific Th1 cells of DBA/2 mouse I-Ed-restricted clones, that express EP3Rs and EP4Rs, was enhanced a maximum of 3-fold by 10(-10) to 10(-8) M PGE2 and 2.5-fold each for the EP1R/EP3R-directed agonist sulprostone (10(-8) and 10(-7) M) and for the EP4R/EP3R/EP2R agonist misoprostol (10(-9) M). Neither PGE2 nor the synthetic analogs affected secretion of IFN-gamma by PMA plus ionomycin-stimulated clones of Th1 cells. Antigen-evoked secretion of IFN-gamma by influenza hemagglutinin-specific mouse lymph node Th1 cells, that also express EP3Rs and EP4Rs, was increased a maximum of 12-fold by 10(-9) to 10(-8) M PGE2, 14-fold by 10(-9) M sulprostone, and 10-fold by 10(-9) M misoprostol. Production of IFN-gamma by either type of Th1 cell was not affected significantly by 10(-6) M PGE2 alone. The generation of IFN-gamma by antigen-stimulated Th1 cells thus is significantly enhanced by physiologically relevant concentrations of PGE2.
[show abstract][hide abstract] ABSTRACT: Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.
Journal of Experimental Medicine 05/1999; 189(7):1111-20. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator in immune tissues that affects many T cell functions through two homologous high-affinity G-protein-coupled receptors, termed VIPR1 and VIPR2. Antigen-stimulated secretion of gamma-interferon (IFN-gamma) by sperm whale myoglobin-specific Th1 cells of DBA/2 mouse I-Ed-restricted clones, which express VIPR1 and VIPR2, was enhanced by 10(-10) M to 10(-7) M VIP. Enhancement of IFN-gamma secretion reached a mean maximum of fourfold for VIP and threefold for a VIPR2-selective agonist, without any effect of a VIPR1-selective agonist. Secretion of IFN-gamma by PMA and ionomycin-stimulated clones of Th1 cells was not altered by VIP. Antigen-stimulated secretion of IFN-gamma by T cell receptor-transgenic, influenza hemagglutinin-specific, and cytokine-differentiated mouse lymph node Th1 cells, which also express VIPR1 and VIPR2, was enhanced by 10(-10) M to 10(-8) M VIP. Enhancement of IFN-gamma secretion increased to a maximum of 14-fold for VIP, 14-fold for the VIPR2-selective agonist, and 20-fold for the VIPR1-selective agonist. In contrast to VIP suppression of interleukin production and lack of effect on IFN-gamma production by T cells stimulated with anti-CD3 antibody or a mitogenic lectin, generation of IFN-gamma by antigen-stimulated T cells is enhanced significantly by physiological concentrations of VIP.
The FASEB Journal 03/1999; 13(2):347-53. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.
Molecular and Cellular Biology 02/1999; 19(1):69-77. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.
[show abstract][hide abstract] ABSTRACT: Linker or adapter proteins provide mechanisms by which receptors can amplify and regulate downstream effector proteins. We describe here the identification of a novel B cell linker protein, termed BLNK, that interfaces the B cell receptor-associated Syk tyrosine kinase with PLCgamma, the Vav guanine nucleotide exchange factor, and the Grb2 and Nck adapter proteins. Tyrosine phosphorylation of BLNK by Syk provides docking sites for these SH2-containing effector molecules that, in turn, permits the phosphorylation and/or activation of their respective signaling pathways. Hence, BLNK represents a central linker protein that bridges the B cell receptor-associated kinases with a multitude of signaling pathways and may regulate the biologic outcomes of B cell function and development.
[show abstract][hide abstract] ABSTRACT: Little is known regarding the diversity of the host T cell response that is required to maintain immunologic control of microbial pathogens. Leishmania major persist as obligate intracellular parasites within macrophages of the mammalian host. Immunity is dependent upon activation of MHC class II-restricted T cells to an effector state capable of restricting growth and dissemination of the organisms. We generated alpha-beta Leishmania-specific (ABLE) TCR transgenic mice with MHC class II-restricted T cells that recognized an immunodominant Leishmania Ag designated LACK. Naive T cells from ABLE mice proliferated in vitro after incubation with recombinant LACK or with Leishmania-parasitized macrophages and in vivo after injection into infected mice. Infected ABLE mice controlled Leishmania infection almost as well as wild-type mice despite a drastic reduction in the T cell repertoire. ABLE mice were crossed to mice with disruption of the TCR constant region alpha gene to create animals with a single alpha beta T cell repertoire. Although mice deficient in all alpha beta T cells (TCR-C alpha 0 mice) failed to control L. major, mice with a monoclonal alpha beta T cell repertoire (ABLE TCR-C alpha 0 mice) displayed substantial control. The immune system is capable of remarkable efficiency even when constrained to recognition of a single epitope from a complex organism.
The Journal of Immunology 01/1998; 160(2):884-9. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Linker or adapter proteins provide mechanisms by which receptors can amplify and regulate downstream effector proteins. We describe here the identification of a novel B cell linker protein, termed BLNK, that interfaces the B cell receptor–associated Syk tyrosine kinase with PLCγ, the Vav guanine nucleotide exchange factor, and the Grb2 and Nck adapter proteins. Tyrosine phosphorylation of BLNK by Syk provides docking sites for these SH2-containing effector molecules that, in turn, permits the phosphorylation and/or activation of their respective signaling pathways. Hence, BLNK represents a central linker protein that bridges the B cell receptor–associated kinases with a multitude of signaling pathways and may regulate the biologic outcomes of B cell function and development.
[show abstract][hide abstract] ABSTRACT: Posttranslational phosphorylation of proteins is an important event in many cellular processes. Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors. Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms. Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction. We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains. From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.